Inhibitory effects of phloridzin dihydrate on the activity of mushroom (Agaricus bisporus) tyrosinase

The inhibitory effects of phloridzin dihydrate on the activity of mushroom tyrosinase have been studied. The results show that phloridzin can inhibit the diphenolase activity of the enzyme and the inhibition displays to be reversible. The IC(50) value was estimated as 110microM. The kinetic analysis showed that the inhibition of phloridzin on the diphenolase activity of the enzyme is of competitive type, and the inhibition constant (K(I)) was determined to be 64.3microM. The inhibitory effects of the different concentrations of phloridzin on the monophenolase activity were also studied. There were almost no changes in the lag period and the steady-state rate, while the plateaus in the inhibitory curve lowered with increasing the concentration of phloridzin when using tyrosine as a substrate.     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) adenocarcinoma cells. The IC₅₀ values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respectively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduction in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

小麦光温敏雄性不育相关基因的DDRT－PCR分析及功能预测

 以小麦光温敏雄性不育系BS210为试材,分别在北京顺义（可育环境）和安徽阜阳（不育环境）两种诱导育性表现的生态环境下种植,并于雌雄蕊分化期、药隔形成期、花粉母细胞形成期、四分体期和单核期5个时期提取小穗组织的RNA,利用DDRT-PCR方法分离不育系在光、温因子诱导下的差异表达mRNA,并通过反向Northern 进行验证.对获得的20条差异表达的EST进行了测序和BLAST分析,得到了4个候选基因的片段,对其进行5′Race 扩增、序列分析及功能预测.结果表明：它们分别与水稻的DNA修复重组蛋白基因rad50、小麦穗部表达的LRR重复序列型类受体激酶基因、玉米叶片坏死斑点L1s1基因的序列相似性分别为89%、89%和88%,另外还筛选到1个未知功能的新基因片段,它和rad50分别在可育和不育环境下表达的mRNA具有相同的5′端编码区,但3′端非编码区     

A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis

Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.     

Relationships between BK virus lineages and human populations

BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney, in which it can cause nephropathy in renal transplant patients. BKV isolates are classified into four subtypes (I-IV) using serological or genotyping methods, and subtype I is further divided into four subgroups, Ia, Ib-1, Ib-2, and Ic, based on DNA sequence variations. To clarify whether there is an association between BK virus lineages and human populations, we examined BKV-positive urine samples collected from immunocompetent individuals at various locations in Europe, Africa, and Asia. Partial BKV DNA sequences (n=299) in these samples were determined and subjected to phylogenetic and single nucleotide polymorphism analysis to classify BKV isolates around the world. The validity of the classification was confirmed by analyses based on complete BKV DNA sequences. Subtype I was the major subtype throughout the studied regions, and subtype IV was prevalent only in Asia and Europe. Subtype-I subgroups showed close relationships to major geographical areas. It has recently been shown that JC virus (a human polyomavirus closely related to BKV) co-evolved with human populations, and the present study thus suggests that host-linked evolution is the general mode of polyomavirus evolution. Additionally, our results indicate certain unique aspects of the relationship between BKV and humans.     

Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca²⁺-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNF, or -amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H₂O₂ or TNF. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca²⁺]_i observed after H₂O₂ or TNF treatment in TRPM2-expressing cells, suggesting that phosphorylation is important in TRPM2 activation. Utilizing a TransSignal PDZ domain array blot to identify proteins which interact with TRPM2, we identified PTPL1 as a potential binding protein. PTPL1 is a widely expressed tyrosine phosphatase, which has a role in cell survival and tumorigenesis. Immunoprecipitation and glutathione-S-transferase pull-down assays confirmed that TRPM2 and PTPL1 interact. To examine the ability of PTPL1 to modulate phosphorylation or activation of TRPM2, PTPL1 was coexpressed with TRPM2 in human embryonic kidney-293T cells. This resulted in significantly reduced TRPM2 tyrosine phosphorylation, and inhibited the rise in [Ca²⁺]_i and the loss of cell viability, which follow H₂O₂ or TNF treatment. Consistent with these findings, reduction in endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a significantly greater rise in [Ca²⁺]_i following H₂O₂ treatment, and enhanced susceptibility to H₂O₂-induced cell death. Endogenous TRPM2 and PTPL1 was associated in U937-ecoR cells, confirming the physiological relevance of this interaction. These data demonstrate that tyrosine phosphorylation of TRPM2 is important in its activation and function and that inhibition of TRPM2 tyrosine phosphorylation reduces Ca²⁺ influx and protects cell viability. They also suggest that modulation of TRPM2 tyrosine phosphorylation is a mechanism through which PTPL1 may mediate resistance to cell death. transient receptor potential channels; oxidative stress     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.